Differential affinity labeling of the two subunits of the homodimeric animal fatty acid synthase allows isolation of heterodimers consisting of subunits that have been independently modified.
To explore the domain interactions that are required for catalytic activity of the multifunctional, homodimeric fatty acid synthase (FAS), we have formulated a strategy that allows isolation of modified dimers containing independently mutated subunits. Either a hexahistidine or a FLAG octapeptide tag was incorporated into the FAS at either the amino terminus, within an internal noncatalytic domain, or at the carboxyl terminus. The presence of the tags had no effect on the activity of the wild-type FAS. His-tagged dimers were mixed with FLAG-tagged dimers, and the subunits were randomized to produce a mixture of His-tagged homodimers, FLAG-tagged homodimers, and doubly tagged heterodimers. The doubly tagged heterodimers could be purified to homogeneity by chromatography on an anti-FLAG immunoaffinity column followed by a metal ion chelating column. This procedure for isolation of FAS heterodimers was utilized to determine whether the two centers for fatty acid synthesis in the FAS dimer can function independently of each other. Doubly tagged heterodimers, consisting of one wild-type subunit and one subunit in which the thioesterase activity had been eliminated, either by mutation or by treatment with phenylmethanesulfonyl fluoride, have 50% of the wild-type thioesterase activity and, in the presence of substrates, accumulate a long chain fatty acyl moiety on the modified subunit, thus blocking further substrate turnover at this center. Nevertheless, the ability of the heterodimer to synthesize fatty acids is also 50% of the wild-type FAS, demonstrating that an individual center for fatty acid synthesis has the same activity when paired with either a functional or nonfunctional catalytic center.
Joshi
A K
AK
Children's Hospital Oakland Research Institute, Oakland, California 94609, USA.
Rangan
V S
VS
Smith
S
S
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D019281:Dimerization; D064429:Fatty Acid Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D000345:Affinity Labels; D005227:Fatty Acids; D011994:Recombinant Proteins; D064429:Fatty Acid Synthases